Effect of 24 Hour Pre‐incubation with Variable Oxygen on Human Islets of Langerhans Function

Abstract

Transplantation of Islets of Langerhans is a form of cell‐based insulin replacement therapy for treatment of Type 1 diabetes. However, this approach requires lifelong immunosuppression, which can lead to severe side effects. Encapsulation of islets (immune‐isolation) within devices has potential to eliminate this critical limitation and open up the use of cell‐based sources, such as stem cell derived beta cells or even porcine islets. The size of the devices create a high density environment which can create hypoxic and ischemic conditions that lead to loss of function and eventually viability of the beta cells. Ultimately, a mechanism for oxygenation is needed to make these devices useful. Therefore, it is important to understand the effect of hypoxic and ischemic conditions on islet viability and function. In this study, we cultured isolated human islets at different oxygen tensions for a period of 24 hours (a time period standard in pre‐transplantation protocols) then examined oxygen consumption rate (OCR) and glucose stimulated insulin secretion (GSIS, using a perifusion system) as a measure of functionality. After isolation, islets were maintain at room air O2 (~ 21%), and were used within 11 days of isolation (11+/−3.8 days, n=12.) Islets were then incubated with variable oxygen for 24 hrs prior to analysis. Following 24 hr incubation, the OCR was 93.7 (+/− 11.05 SEM), 135.2 (+/− 7.0), 125.5 (+/− 16.0) and 92.7 (+/− 7.7) (nmol O2/min*mg DNA) for islets incubated in 1%, 21%, 40% and 95% oxygen, respectively. Therefore, basal OCR was clearly influenced by ambient oxygen with 21% O2 showing the highest OCR with lowest variability. For islets pre‐incubated with 21% O2, the basal insulin secretion and GSIS (20 min stimulation) rates were 2.0 +/− 0.7 and 13.6 +/− 3.7 (pg insulin/20 min*ng DNA), respectively. The basal secretion rate for islets maintained at 1% O2 (4.5 +/− 0.34) was significantly elevated compared to islets maintained at 21%. Basal secretion rates for 40% and 95% were unchanged compared to 21% O2. A standard approach to evaluate relative insulin secretion is to calculate what is termed a stimulation index (SI). The SI is calculated as the area under the curve for glucose stimulated secretion minus basal secretion then divided by the basal insulin secretion rate. For islets pre‐incubated with 21% O2, the SI was 6.4 +/− 1.3. There was no significant difference for islets incubated in 40% O2 (7.2 +/− 0.4), but islets incubated in 1% and 95% exhibited significant reductions in SI by 50% (3.2 +/− 1.1) and 34% (4.2 +/− 1.1), respectively. These findings indicate that normal to slightly elevated oxygen must be maintained in order to promote optimal islet function acutely. Furthermore, the obvious relation between ambient O2 and function must be considered in the context of islet/β‐cell maintenance within in vivo devices. This is particularly pertinent, since we've shown that changes in HIF‐1α and gene expression occur in association with 24 hrs of hypoxia in adult porcine islets.Support or Funding InformationThis work was supported by: JDRF Grant: 2‐SRA‐2014‐289‐Q‐R and NIH/NIDDK DP3 Grant: 1DP3DK106933‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.